The purpose of this research is to develop methodology that enhances the usefulness of nitroxide radical spin-labels as probes of structure and function of biological systems using electron paramagnetic resonance (EPR) spectroscopy. By "methodology" we mean (1) instrument development, (2) techniques for the use of instruments including computer data processing, (3) theoretical analysis of the basis for the techniques, (4) measurements of physcial properties of nitroxides, and (5) the carrying out of prototype applications of the new methods. The problems that interest us are (1) tightness of binding of labels to proteins. Tighter binding enhances the usefulness of Saturation-Transfer Spectroscopy, and measurements of binding tightness may yield informaton on conformational fluctuations of proteins. (2) Analysis of jump motions and anisotropic motions in all relevant time scales, but particularly at longer times. (3) Spin-lattice relaxation mechanisms of nitroxide, particularly when undergoing very slow motions. And (4) Dynamic processes in model membranes under non-equilibrium conditions. The techniques we will use include (1) Saturation Recovery, (2) Multi- and Differential Frequency spectroscopy, (3) Spin-probe--spin-label methods, (4) ELDOR, (5) Saturation-Transfer spectroscopy, and (6) Stop-flow. Many of our approaches involve time-domain EPR spectroscopy, which we view as the central thrust of EPR development in the next decade.